The eukaryotic type IB DNA topoisomerase family includes the nuclear topo I and the topoisomerases encoded by vaccinia and other cytoplasmic poxviruses. The type IB enzymes relax DNA supercoils by breaking and rejoining one strand of the DNA duplex. DNA strand scission occurs via transesterification and results in the formation of a covalent DNA-(3'- phosphotyrosyl)-Topo intermediate. The participation of type IB topoisomerases in DNA replication, genetic recombination, and transcription plus the fact that nuclear topo I is the target of the campothecin anti-tumor drugs, mandates a thorough understanding of their mechanism of action. This laboratory uses vaccinia virus as a model system to study topo IB. The 314-amino acid vaccinia enzyme is the smallest topo known. A distinctive feature of the pox-virus enzyme is its sequence specificity in strand cleavage. Vaccinia topo binds forms a covalent adduct at sites containing a pentapyrimidine element 5'(C/T)CCTT in the scissile strand. Our long term goals are to: (i) elucidate the structural basis for DNA transesterification chemistry and CCCTT target site recognition, (ii) define the catalytic repertoire of topo IB, especially with respect to recombination reactions, and (iii) dissect the essential role of topoisomerase during the vaccinia replicative cycle. Five specific aims are proposed herein to advance this agenda. (1) Identification o fall catalytic moieties on the enzyme by comprehensive mutagenesis and biochemical characterization of the mutant proteins. (2) Determination via -ray crystallography of the molecular structure of vaccinia topoisomerase bound to its recognition element CCCTT in duplex DNA. (3) Kinetic analysis of the contribution of individual DNA phosphates and bases to target site recognition and transesterification. (4) Analysis of topoisomerase-catalyzed DNA rearrangements in vitro. (5) Construction of a mammalian host cell line expressing vaccinia topoisomerase that will permit the isolation and propagation of a mutant vaccinia virus deleted for the topoisomerase gene. With the genetic system established, it will be possible to determine which steps in vaccinia replication are affected during synchronous infection of on- permissive cells with the deltatopo virus.